Molecular Evolution and Increasing Macrolide Resistance of Bordetella pertussis, Shanghai, China, 2016–2022

Resurgence and spread of macrolide-resistant Bordetella pertussis (MRBP) threaten global public health. We collected 283 B. pertussis isolates during 2016–2022 in Shanghai, China, and conducted 23S rRNA gene A2047G mutation detection, multilocus variable-number tandem-repeat analysis, and virulence genotyping analysis. We performed whole-genome sequencing on representative strains. We detected pertussis primarily in infants (0–1 years of age) before 2020 and older children (>5–10 years of age) after 2020. The major genotypes were ptxP1/prn1/fhaB3/ptxA1/ptxC1/fim2–1/fim3–1 (48.7%) and ptxP3/prn2/fhaB1/ptxA1/ptxC2/fim2-1/fim3-1 (47.7%). MRBP increased remarkably from 2016 (36.4%) to 2022 (97.2%). All MRBPs before 2020 harbored ptxP1, and 51.4% belonged to multilocus variable-number tandem-repeat analysis type (MT) 195, whereas ptxP3-MRBP increased from 0% before 2020 to 66.7% after 2020, and all belonged to MT28. MT28 ptxP3-MRBP emerged only after 2020 and replaced the resident MT195 ptxP1-MRBP, revealing that 2020 was a watershed in the transformation of MRBP.

Despite the variation in virulence genotypes in circulating strains, different B. pertussis subtypes are prevalent in the world.The multilocus variablenumber tandem-repeat analysis (MLVA) type (MT) 27 strain carrying the genotype of ptxP3/ptxA1/prn2/ fm3-1 has become the predominant B. pertussis strain in many countries (22).However, MT27 has seldomly been reported in China, whereas the MT55, MT195, or MT104 strains harboring the ptxP1 allele have been reported to circulate in some regions of China (23,24).Macrolide-resistant B. pertussis (MRBP), which carries an A-to-G transition at nucleotide position 2047 (A2047G mutation) in a region critical for erythromycin binding, emerged in some countries, but was only frequently detected in China (15,16,(25)(26)(27).MRBPs generally carry ptxP1 and fhaB3, but 2 novel MRBPs belonging to MT28 and MT27 carrying ptxP3 and fhaB1 were reported in mainland China (15,28).
Our recent study reported that MT28 ptxP3-MRBP has emerged and spread in Shanghai, China, during 2021-2022 (29).However, several urgent questions remain to be resolved.For example, was ptxP3-MT28 MRBP dominant in Shanghai in the long term, or did it emerge in 2021 and 2022?Why and when did ptxP3-MT28 MRBP emerge in Shanghai, and how did they evolve?To resolve those questions, we conducted further research during 2016-2022 to reveal the evolution of MRBP in Shanghai.We collected a total of 283 B. pertussis isolates during 2016-2022 in Shanghai and systematically analyzed the antimicrobial resistance and molecular evolution of those strains.

Enrollment of Case-Patients with B. pertussis Infection
We included in the study a total of 1,065 children admitted to the Children's Hospital of Fudan University and diagnosed with pertussis during January 2016-October 2022, who had nasopharyngeal swab (NP) samples collected and delivered to the microbiology laboratory for B. pertussis culture, antimicrobial resistance testing, and PCR detection.We extracted DNA from NP samples and performed real-time PCR (LightCycler 480; Roche, https://www.roche.com) to detect nucleic acids according to the protocol of a pertussis bacteria nucleic acid detection kit based on the PCR-fluorescent probe method (Yilifang Biotechnology, http://www.yilifangbio.com).The laboratory testing results and data collection were based on electronic medical records during hospitalization or clinic visits, and all data analysis was anonymous.The study protocol was approved by the Ethics Committee of the Children's Hospital of Fudan University (approval no.2022-66).

PCR and Sequencing for 23S rRNA A2047G Mutation Detection and Virulence Genotyping Analysis
We obtained 692 B. pertussis strains in 2016 (11 strains), 2017 (177 strains), 2018 (165 strains), 2019 (169 strains), 2020 (1 strain), 2021 (30 strains), and 2022 (139 strains).Because very few strains were obtained from 2016, 2020, and 2021, we selected all 42 strains for this study.We chose other isolates by the systematic sampling method, yielding 50 strains in 2017, 45 strains in 2018, 74 strains in 2019, and 72 strains in 2022.We gave each strain a number and then chose it by a random method to ensure each strain had an equal chance of being chosen through the use of an unbiased selection method.We selected a total of 283 isolates for further analysis.

MLVA
We performed MLVA by following the procedures described by Schouls et al. (32).We amplified 5 loci (variable-number tandem-repeat [VNTR] 1, VN-TR3a/VNTR3b, VNTR4, VNTR5, and VNTR6) by using PCR detection.We calculated the number of repeats at each VNTR locus from the DNA fragment length.We assigned an MT on the basis of the combination of repeat counts for VNTRs 1, 3a, 3b, 4, 5, and 6, as described in previous reports (15,32).

Public Genome Dataset
We included a total of 1,491 public genomes of B. pertussis strains from China (15,21,28) ).We downloaded raw short-read sequencing data from the National Center for Biotechnology Information Sequence Read Archive (https:// www.ncbi.nlm.nih.gov/sra).We filtered sequencing reads by using Trimmomatic (34), and we performed de novo genome assembly of public data by using SPAdes (35) with default settings.

Single-Nucleotide Polymorphism Calling and Phylogenetic Analysis
We identified core-genome (regions present in >99% of isolates) single-nucleotide polymorphisms (SNPs), as previously described (36).In brief, we aligned the assemblies against the reference genome (Gen-Bank accession no.NC_002929.2,Tohama I) by using MUMmer (37) to generate whole-genome alignment.We performed SNP calling by using SNP-Sites (38) on the basis of the alignment.We identified the repetitive regions of the reference genome by using Tandem repeats finder and self-aligning by blastn (https://blast.ncbi.nlm.nih.gov).We excluded SNPs located in repetitive regions from further analysis.We constructed a maximum-likelihood phylogenetic tree by using RAxML-NG (39) under the general time-reversible with gamma distribution model.

Statistical Analysis
We analyzed data by using the t test, χ 2 test, or Fisher exact test, as appropriate.We performed all statistical analyses by using the SPSS Statistics 13.0 (IBM, https://www.ibm.com).We considered p<0.05 to be statistically significant.

Discussion
In this study, we systematically investigated the clinical characteristics, antimicrobial resistance profiles, and molecular evolution of B. pertussis strains in Shanghai, China, during 2016-2022.Pertussis was primarily diagnosed in infants before 2020 but mostly in older children and adolescents after 2020.MRBPs remarkably increased, from 36.4% in 2016 to 97.2% in 2022.MT28 ptxP3/prn2/fhaB1-MRBP emerged only after 2020 and replaced MT195 ptxP1/prn1/fhaB3-MRBP, which was prevalent before 2020, indicating that 2020 was a watershed in the transformation of MRBP in Shanghai, China.
MRBP strains were widely prevalent in western China and mainly linked to MT195, MT104, and MT55 (26).Wu et al. (15) showed that MT28 MRBP with genotype of ptxP3/fhaB1/prn9 was first identified in Anhui Province, China, revealing the emergence of ptxP3-MRBP in mainland China (15).In this study, the circulating B. pertussis strains changed greatly from 2016 to 2022.MT195 presented the VNTR profiles of 8-6-0-7-6-8, whereas MT28 showed the profiles of 8-7-0-7-6-8, and MT27 showed the profiles of 8-7-0-7-6-7.Although those subtypes have minor differences on VNTR3a or VNTR6, their virulence genotypes and A2047G mutation carriages  were quite different, making the circulating strains very heterologous.All MRBPs before 2020 harbored ptxP1 and 51.4% belonged to MT195, whereas ptxP3-MRBP, which was absent before 2020, increased to 66.7% after 2020, and all belonged to MT28.WGS analysis further revealed that MT28-MRBP was quite heterologous with MT195-MRBP, revealing the different molecular characteristics of MRBP prevalent before and after 2020 in Shanghai.MT28-MRBP in this study was quite different from the international strains but represented close relevance to MT28-MSBP isolated before 2020, which indicates that MT28-MRBP was not reported from other countries but more likely because the resident MT28-MSBP acquired the A2047G mutation and became resistant to macrolides.Moreover, the emergence and spread of MT28 ptxP3-MRBP in Shanghai were probably related to the selection pressure from high usage of macrolides and vaccination.Macrolides were excessively used for treating pertussis, which might participate in the selection of ptxP3-MRBP.
Of interest, although MRBPs are highly resistant to macrolides, most (60.1%) of the MRBP patients were still treated with macrolides in this study.In addition, compared with vaccine strains in China with the genotype of ptxP1/fhaB1/prn1/ptxA2/ptxC1, MT28 harbored more gene variants, including ptxP3, prn2, ptxA1, and ptxC2 than MT195, which carried fhaB3 and ptxA1.Currently, 2 types of diphtheria, tetanus, and pertussis (DTaP) vaccine formulations are licensed in China: one is the 2-component DTaP vaccine containing PT and FHA, another is the 3-component DTaP vaccine containing PT, FHA, and PRN (43).The circulating B. pertussis has evolved, mainly changed from ptxP1 to ptxP3 lineage, indicating the ptxP3 variation reflect selective advantage under high coverage with acellular pertussis vaccine (42).Previous study showed that prn2 variation affected the efficacy of commercial vaccine, and mice studies suggested that the incorporation of prn2 to vaccine could enhance the ACV's efficacy (44).Moreover, studies from Safarchi et al. (45) and Van Gent et al. (46) demonstrate that ptxP3/ prn2-BP colonized better than the ptxP1/prn3-BP strain and provide the evidence for increased fitness and better immune evasion of ptxP3/prn2 strains in a mouse model involving mice immunized with 3-component ACVs.Therefore, we hypothesized that prn2 and ptxP3 variation in MT28 strains may play a role in better fitness and immune evasion compared with ACVs in China, causing MT28-BP to be selected by the vaccination and then to spread quickly.The exact relationship between prn2/ptxP3 variant and vaccine escape needs further study.
In this study, pertussis was primarily detected in infants before 2020 but was mostly detected in older children after 2020.We propose 2 potential hypotheses for this age shift.First, the age shift was closely related to the emergence of MT28-MRBP; ptxP3/ptxA1/ptxC2/prn2carrying MT28 strains, which emerged and spread after 2020 could avoid the immunity of vaccine and weaken the vaccine effects, making the pertussis populations shift from unvaccinated or incompletely vaccinated infants to vaccinated population.Second, the COVID-19 pandemic increased the public awareness of microbiologic laboratory testing in children with respiratory symptoms, so more older children who were not considered as the primary pertussis population before 2020 accepted B. pertussis testing and were diagnosed with pertussis after 2020, making 2020 become the watershed moment for the shift of pertussis population.
In conclusion, we systematically investigated the molecular evolution of MRBPs to clarify the evolution of MRBP from MT195 to MT28 in Shanghai, China, during 2016-2022, revealing that 2020 was watershed in the transformation of MRBPs from MT195 ptxP1/ prn1/fhaB3-alleles to MT28 ptxP3/prn2/fhaB1-alleles in Shanghai.The emergence and spread of MT28 ptxP3-MRBP strains are likely attributable to the A2047G mutation and the selection pressure from vaccination and high usage of macrolides, which will further complicate the epidemiology of pertussis and evolve to pose a looming threat to global public health.Therefore, worldwide surveillance of the molecular evolution and AMR profiles of circulating B. pertussis, especially ptxP3-MRBP, is urgent.

Figure 1 .
Figure 1.Distributions of pertussis patients in different age groups, Shanghai, China, 2016-2022.Pertussis was primarily detected from infants (0-1 years of age) before 2020 but mostly from older children and adolescents (>5-10 years of age) after 2020.

Figure 4 .
Figure 4. Minimum-spanning tree of 283 Bordetella pertussis MTs, Shanghai, China, 2016-2022.A) Virulence genotype profiles; B) A2047G mutations; C) pre-2020 versus post-2020.Circle sizes indicate the number of each MT.Differences in the length and thickness of the lines linking 2 circles indicate differences in the number of variable-number tandem repeats between the 2 linked MTs.MT, multilocus variable-number tandem repeat analysis type.
This study was funded by the National Key Research and Development Program of China (grant nos.2021YFC2701800 and 2021YFC2701801), the Key Development Program of the Children's Hospital at Fudan University (grant no.EK2022ZX05), and the National Natural Science Foundation of China (grant no.82202567).Author contribution: C.W., X.Z., and P.F.designed the experiments and revised the manuscript.C.Y. performed WGS analysis, analyzed the data, and revised the manuscript.P.F.analyzed the data and wrote the manuscript.J.Z. performed most of the experiments.G.L. performed experiments guidance.Y.N., G.Y., and L.Z. participated in the experiments.